The major objective of our work is to advance the scientific principles governing vector biology that will result in improved gene transfer/genome editing approaches. All of the studies described in this proposal have the potential to enhance gene therapeutics for the treatment of Hemophilias A and B. However, the work outlined in the proposal is a platform technology that will advance gene therapeutics/genome editing. The ultimate goal will be to provide a curative treatment after a single vector dose at the time of diagnosis. This will be important not only for treating hemophilia but other inherited metabolic diseases. Specifically, we propose studies that will further enhance nuclease-free AAV mediated homologous recombination (AAV-HR) strategies. We will determine how factors that include host gene selection, composition of chimeric mRNA transcripts, tissue regeneration, transcription, chromatin, R-loop, and direction and timing of DNA replication affect the efficiency of AAV-HR and AAV-mediated HR transgene expression. We will also pursue preclinical strategies for temporarily knocking down genes encoding proteins in the FANCM protein complex. Furthermore, we study FDA approved drugs, which inhibit the ribonucleotide reductase complex and may enhance AAV-HR in the liver. We will test a novel rAAV capsid variant we previously discovered, which has the capacity to package larger genomes for its ability to package more robust classical AAV-human factor VIII expression cassettes and allow for AAV-HR of the hFVIII coding sequence. The work outlined will not only address mechanistic aspects related to AAV-HR but it will expand the utility of this approach and define potential limits to the current technology.